Cloning of the Zucchini Opiate Receptor

by Lloyd
Fricker, Albert Einstein College of Medicine, Bronx, New York

This paper describes the cloning of the zucchini opiate receptor using
an expression assay. We have undertaken this project for the following reasons:

1) Opiate receptors are important.
2) Cloning things is important.
3) Therefore, cloning the opiate receptor must be very important.

Our model system involves the zucchini, which avoids many of the ethical
problems associated with using small defenseless animals for research, especially
for studies involving pain. Vegetables are also cheaper than laboratory
animals, and they taste better as well (1). We have previously demonstrated
that zucchini contain functional receptors for opiates (2), so this was
the vegetable of choice.

Materials and Methods

Zucchini were obtained from a local breeding facility and were transported
back to the laboratory on ice. They were housed in the crisper section of
the refrigerator until use. Mr. Potato Head was obtained from Toys-B-Us
and assembled with the help of a 5 year old. All procedures were approved
by the Institutional Squash Use Committee (ISUC).

Results and Discussion

When zucchini were subjected to the standard hot-plate test, they showed
a normal jumping reflex (Figure 1, Panel A). Much to the demise of the unfortunate
vegetable, injection of 10 mg/kg of morphine into the tail stem of the zucchini
caused a dramatic decrease in the perception of pain, as well as instilling
a false sense of serenity to the vegged-out zucchini (Figure 1, Panel B).
Injection of 10 mg/kg naloxone (an opiate antagonist) rudely awakened the
blissful vegetable to the cruel reality of the situation, with full restoration
of the jumping reflex (Figure 1, Panel C).

Figure 1. The effect of morphine and naloxone on the hot plate induced
jumping reflex in a zucchini. A: the normal jumping reflex demonstrated
when an adult female Burpee brand zucchini is placed on a scalding hot surface,
such as a Therm-O-Swirl that had been left on for several days. B: Injection
of 10 mg/kg of morphine into the tail stem of the zucchini eliminates the
jumping reflex. C: Injection of 10 mg/kg naloxone (an opiate antagonist)
into the morphine-treated zucchini restores the jumping reflex.

In a search for an organism that has a jumping reflex but which is not
responsive to morphine, we tested a variety of fruits and vegetables. Some,
like milk, did not show a jumping reflex at all, while others (bananas,
popcorn) showed a robust morphine-sensitive jumping reflex. During this
screening, we found that potatoes showed a jumping reflex that was not responsive
to morphine (not shown). To facilitate the expression cloning, we tested
the highly expressive Mr. Potato Head, which is evolutionarily related to
the Idaho potato (3). As shown in Figure 2, Mr. Potato Head showed a jumping
reflex when tossed onto the same scalding hot surface as used for the zoned
out zucchini in Figure 1.

A hefty dose of morphine did nothing to alter the jumping reflex of Mr.
Potato Head, nor did it appear to alter the expression of fear caused by
the hot surface (Figure 2, Panel B). Injection of 10 mg/kg of naloxone also
had no influence on the jumping reflex of Mr. Potato Head (Figure 2, Panel
C).

Figure 2. The effect of morphine and naloxone on the hot plate induced
jumping reflex in Mr. Potato Head. A: The normal jumping reflex when an
adult male Mr. Potato Head (obtained from Toys-B-Us). B: Injection of 10
mg/kg of morphine i.p. (intra-potato). C: Injection of 10 mg/kg of naloxone
i.p. into the morphine-treated toy.

Taken together, these results indicate that Mr. Potato Head does not contain
functional opiate receptors.

To clone the zucchini opiate receptor, we first created a zucchini cDNA
library from RNA (actually, zRNA) isolated from the neuroinfundibular lateral
geniculate moosel structure of the zucchini, which has been previously shown
by autoradiography to contain large amounts of opiate receptors (2). The
library was then made by doing all the things that one does when one makes
a library. A portion of the library is shown in Figure 3.

Figure 3. Standard zucchini library (volumes 62-65).

The zucchini library was then transfected into Mr. Potato Head using a
modification of the calcium phosphate method (the modification used NaCl
in place of calcium phosphate, and ketchup in place of the glycerol). The
zucchini/Mr. Potato Head library shows partial phenotype of both parent
organisms, and tastes great either raw or deep fried. A portion of the library
is shown in Figure 4.

Figure 4. Zucchini/Mr. Potato Head expression library.

The zucchini/Mr. Potato Head library was screened using an expression assay.
Specifically, the change in "expression" upon painful stimulus,
and the ability of morphine to attenuate this change, was tested for each
clone. One positive clone was identified which did not respond to an extremely
painful stimulus in the presence of morphine (Figure 5).

Figure 5. Representative (actually, the only) clone from the zucchini/Mr.
Potato Head library which does not show a change in expression when subjected
to a painful stimulus under the influence of morphine.

To confirm the morphine-induced insensitivity to pain, the unfortunate
clone was dosed with 10 mg/kg morphine and then subjected to the blistering
hot surface of the Therm-O-Swirl. In the presence of morphine, the poor
clone lost all signs of a jumping reflex and is shown moments before bursting
into flames (Figure 6).

To eliminate the possibility that this clone has simply lost the jumping
reflex, rather than gaining the opiate receptor, the clone was tested in
the absence of morphine. Without the drug, the zucchini/Mr. Potato Head
clone showed a normal jumping reflex on the hot plate test (Figure 7, Panel
A). Note the expression of fear on the clone. With 10 mg/kg morphine coursing
through its veins, the blissed-out clone lost all concept of fear of hot
surfaces, and did not display any survival instincts to escape from the
hot surface (Figure 7, Panel B). Injection of naloxone fully restored the
jumping reflex, and the apparent feeling of pain to the clone (Figure 7,
Panel C). This is one unhappy clone!

Figure 7. Panel A: This is a clone. Panel B: This is a clone on drugs
(10 mg/kg morphine). Panel C: This is a clone on even more drugs (10 mg/kg
naloxone, in addition to the morphine). Any questions?

The cDNA from the zucchini/Mr. Potato Head clone was isolated and sequenced,
but since the actual order of the nucleotides is of interest only to our
competitors, we have not shown the sequence. Go clone it yourself if you
really want to know. We're holding this confidential until we get it published
in a good journal, like Cell, Science, or Better Homes and Gardens.

Acknowledgments

Thanks to Lakshmi, Mike, Mark, Bruce, and Jim for helping arrange and photograph
the zucchini late one night in the Herbert lab. This work was previously
presented as a poster at a Society for Neuroscience meeting in 1986, and
is also to be included in an upcoming book to be published by HMS Beagle.